ABSTRACT Conjunctivitis (pink eye) inflames the inner surface of the eyelid and sclera, and affects ~6 million Americans annually. Symptoms include hyperemia, edema, watery discharge, itching and burning. Adenovirus (AdV) infections account for ~70% of infectious conjunctivitis. AdV is highly contagious, spreads rapidly to the contralateral eye and to unaffected family members and co-workers. In the US, AdV Conjunctivitis causes >3 million missed school days per year with concomitant missed days of work for parents There are no FDA approved treatments for Adenoviral Conjunctivitis and $430M/yr is wasted on ineffective antibiotic therapy. RNA Interference (RNAi) responses have great potential to target the entire Adenovirus genome, including critical genes required for production of infectious virus, such as the L3 Protease. The goal of this project is to treat preclinical models of ocular adenovirus with our next-generation RNAi prodrug, called RiboNucleic Neutrals (siRNNs). Unlike siRNAs, siRNNs have many drug-like properties of extreme stability and deliverability. Importantly, RNAi responses last for >one month, which means patients and their families would only a require a single dose. We hypothesize that selective ocular RNAi knockdown of the AdV L3 gene (Protease, pVI, Hexon) will prevent AdV infectious particle maturation, thereby treating and preventing AdV EKC, with an acceptable efficacy/toxicity ratio to treat the infected eye and prevent spread to the contralateral eye with a safety profile that permits its use in children and prophylactically in uninfected individuals. Aim 1: Optimize siRNN Topical Delivery in Mice Expressing Ocular Luciferase We will optimize L3 siRNN RNAi triggers targeting the critical L3 gene in Adenovirus that encodes the Protease and delivery into the ocular epithelium. Aim 2: Treat Adenovirus Rabbit Models with siRNNs Targeting Adenovirus L3 Gene Using L3 siRNN RNAi triggers, we will perform a Prevention study and an Antiviral Efficacy study in rabbit models to inhibit production and spread of infectious Adenovirus. Our overarching goal is to use the data generated by this study to move the next-gen L3 TAT-siRNN RNAi trigger forward towards declaring a clinical candidate for IND-enabling studies.